SUMMARY Prevalent congenital diseases, including the Di George/22q11DS, Noonan and related syndromes, present with both cardiac defects and craniofacial dysmorphism. The 22q11 Deletion Syndrome arises from eponymous deletions that cause TBX1 haploinsufficiency, while Noonan and related syndromes are RASopathies that affect the RAS-MAPK signaling pathway. However, understanding the etiology of combined cardio-craniofacial defects requires insights into the cellular and developmental contexts of gene function. In early amniote embryos, the second heart field (SHF) and branchiomeric/pharyngeal head muscles emerge from a common population of multipotent progenitors in the cardiopharyngeal mesoderm. TBX1 is thought function in cardiopharyngeal progenitors, to control both pharyngeal myogenesis and SHF development, and interact with Fibroblast Growth Factor (FGF)-MAPK signaling during heart development, highlighting the importance of studying Tbx1 and FGF-MAPK signaling in the cellular context of early cardiopharyngeal development. The tunicate Ciona emerged as a simple and powerful chordate model to study early cardiopharyngeal development, with high spatial and temporal resolution. In Ciona, four multipotent cardiopharyngeal progenitors undergo stereotyped migration and cell divisions, producing distinct first and second cardiac, and pharyngeal muscle lineages that deploy gene networks conserved with vertebrates. Leveraging the unique strengths of the Ciona system, and extensive previous work using lineage-specific perturbations, including CRISPR/Cas9-mediated mutagenesis, quantitative imaging, and multiplexed single cell genomics methods, this proposal will first explore the establishment of spatial patterns. The proposed work will address how a dynamic cardiopharyngeal niche helps polarize MAPK signaling and Tbx1/10 activation; how an intrinsic determinant of mitotic spindle positioning, the RhoGAP Depdc1, helps progenitors orient their divisions with regards to the niche, and analyze the molecular basis for the antagonism between MAPK signaling and the early heart program. Second, this proposal will explore the temporal dynamics underlying transitions between cardiopharyngeal states, by studying how de novo gene expression and fate choices are coupled with cell divisions, and how transcriptome changes in maturing progenitors determine the competence of cardiopharyngeal progenitors to form heart and pharyngeal muscle precursors. Completion of this ambitious proposal will yield far-reaching insights into emerging concepts of broad significance for cardiovascular developmental and stem cell biology.